Butyrate, neuroepigenetics and the gut microbiome: Can a high fiber diet improve brain health?
As interest in the gut microbiome has grown in recent years, attention has turned to the impact of our diet on our brain. The benefits of a high fiber diet in the colon have been well documented in epidemiological studies, but its potential impact on the brain has largely been understudied. Here, we will review evidence that butyrate, a short-chain fatty acid (SCFA) produced by bacterial fermentation of fiber in the colon, can improve brain health. Butyrate has been extensively studied as a histone deacetylase (HDAC) inhibitor but also functions as a ligand for a subset of G protein-coupled receptors and as an energy metabolite. These diverse modes of action make it well suited for solving the wide array of imbalances frequently encountered in neurological disorders. In this review, we will integrate evidence from the disparate fields of gastroenterology and neuroscience to hypothesize that the metabolism of a high fiber diet in the gut can alter gene expression in the brain to prevent neurodegeneration and promote regeneration.


Interaction of the enantiomers of fluoxetine and norfluoxetine with human liver cytochromes P450.
(R)- and (S)-fluoxetine were found to be competitive inhibitors of P450 2D6-mediated bufuralol 1'-hydroxylation in vitro, yielding Ki values of 1.38 +/- 0.48 and 0.22 +/- 0.11 microM, respectively. Their N-demethylated metabolites were also found to be potent inhibitors (Ki, (R)-norfluoxetine, 1.48 +/- 0.27 microM; (S)-norfluoxetine, 0.31 +/- 0.04 microM). The microsomal (R)- and (S)-fluoxetine N-demethylase activities for 14 human liver samples were on average 29.6 +/- 13.5 and 19.4 +/- 11.8 pmol of product/min/mg of protein, respectively. The individual rates of N-demethylation correlated with microsomal immunodetectable P450 2D6 levels; (R)-fluoxetine, r = 0.64, P < .05; (S)-fluoxetine, r = 0.63, P < .05. However, this correlation was significantly weaker than the excellent correlation obtained for P450 2D6-marker bufuralol 1'-hydroxylase activity and P450 2D6 levels (r = 0.92, P < or = .01). Quinidine, a potent inhibitor of P450 2D6, inhibited the demethylation of each enantiomer by only approximately 20% at a concentration 300 times greater than the Ki determined for the quinidine inhibition of bufuralol 1'-hydroxylase. Furthermore, antiserum recognizing P450 2D6 inhibited 82% of microsomal bufuralol 1'-hydroxylase activity but only 27% of the (R)-fluoxetine N-demethylase activity in the same human liver sample. In summary, these data indicate that the enantiomers of fluoxetine and norfluoxetine are potent inhibitors of P450 2D6 and that P450 forms other than P450 2D6 appear to be responsible for the majority of microsomal fluoxetine N-demethylation.


Allosteric modulation of proteinase 3 activity by anti-neutrophil cytoplasmic antibodies in granulomatosis with polyangiitis.
Anti-neutrophil cytoplasmic antibodies (ANCA) with proteinase 3 (PR3) specificity are a useful laboratory biomarker for the diagnosis of Granulomatosis with Polyangiitis (GPA) and are believed to be implicated in the pathogenesis. It has been repeatedly suggested that disease activity of GPA is more closely related to the appearance and rise of PR3-inhibiting ANCA than to an increase of total ANCA. Previous studies on a limited number of patient samples, however, have yielded inconclusive results. To overcome the previous methodological limitations, we established a new ultrasensitive method to quantify the inhibitory capacity of PR3-ANCA using small volumes of plasma from patients with GPA. A large collection of longitudinally-collected samples from the Wegener Granulomatosis Etanercept Trial (WGET) became available to us to determine the functional effects of ANCA on PR3 in comparison to clinical disease manifestations. In these patient samples we not only detected PR3-ANCA with inhibitory capacity, but also PR3-ANCA with enhancing effects on PR3 activity. However no correlation of these activity-modulating PR3-ANCA with disease activity at either the time of enrollment or over the course of disease was found. Only patients with pulmonary involvement, especially patients with nodule formation in the respiratory tract, showed a slight, but not significant, decrease of inhibitory capacity. Epitope mapping of the activity-modulating PR3-ANCA revealed a binding on the active site surface of PR3. Yet these ANCA were able to bind to PR3 with an occupied active site cleft, indicating an allosteric mechanism of inhibition. The recently described signal ratio between the MCPR3-3 and MCPR3-2 capture ELISA was consistent with the binding of activity-modulating ANCA to the active site surface. Evidence for a shared epitope between activity-modulating PR3-ANCA and MCPR3-7, however, was very limited, suggesting that a majority of PR3-ANCA species do not inhibit PR3 by the same mechanism as previously reported for MCPR3-7.


Subtypes of voltage-sensitive calcium channels in cultured rat brain neurons.
Subtypes of voltage-sensitive calcium channels have been investigated in cultured rat brain neurons using two classes of specific probes, dihydropyridine compounds and omega-conotoxin. Membranes prepared from cultured neurons contain specific binding sites for [3H]PN200-110, a dihydropyridine antagonist, and for 125I-omega-conotoxin with a stoichiometry of about 1:1. A depolarization induced 45Ca2+ influx into intact brain neurons was partially inhibited by a dihydropyridine antagonist, nifedipine and stimulated by a dihydropyridine agonist, Bay K8644. This dihydropyridine sensitive 45Ca2+ flux was insensitive to omega-conotoxin at concentrations which saturate the specific toxin binding sites indicating that in cultured brain neurons, dihydropyridine-sensitive calcium channels are not sensitive to omega-conotoxin.


Intermittent hypoxia degrades HIF-2alpha via calpains resulting in oxidative stress: implications for recurrent apnea-induced morbidities.
Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O(2). A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2alpha is an orthologue of HIF-1alpha, we examined the effects of IH on HIF-2alpha, the O(2)-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1alpha, HIF-2alpha was down-regulated by IH. Similar down-regulation of HIF-2alpha was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2alpha whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2alpha degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2alpha prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2alpha degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2alpha via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.


The first genome sequences of human bocaviruses from Vietnam.
As part of an ongoing effort to generate complete genome sequences of hand, foot and mouth disease-causing enteroviruses directly from clinical specimens, two complete coding sequences and two partial genomic sequences of human bocavirus 1 (n=3) and 2 (n=1) were co-amplified and sequenced, representing the first genome sequences of human bocaviruses from Vietnam. The sequences may aid future study aiming at understanding the evolution of the pathogen.


Genetic code redundancy and its influence on the encoded polypeptides.
The genetic code is said to be redundant in that the same amino acid residue can be encoded by multiple, so-called synonymous, codons. If all properties of synonymous codons were entirely equivalent, one would expect that they would be equally distributed along protein coding sequences. However, many studies over the last three decades have demonstrated that their distribution is not entirely random. It has been postulated that certain codons may be translated by the ribosome faster than others and thus their non-random distribution dictates how fast the ribosome moves along particular segments of the mRNA. The reasons behind such segmental variability in the rates of protein synthesis, and thus polypeptide emergence from the ribosome, have been explored by theoretical and experimental approaches. Predictions of the relative rates at which particular codons are translated and their impact on the nascent chain have not arrived at unequivocal conclusions. This is probably due, at least in part, to variation in the basis for classification of codons as "fast" or "slow", as well as variability in the number and types of genes and proteins analyzed. Recent methodological advances have allowed nucleotide-resolution studies of ribosome residency times in entire transcriptomes, which confirm the non-uniform movement of ribosomes along mRNAs and shed light on the actual determinants of rate control. Moreover, experiments have begun to emerge that systematically examine the influence of variations in ribosomal movement and the fate of the emerging polypeptide chain.


The Epidemiology of Depressive Symptoms in the Last Year of Life.
Depression impacts quality of life at all life stages, but the epidemiology of depression in the last year of life is unknown. This study's objectives were to document the epidemiology of depressive symptoms in the year prior to death and to assess how the trajectory of depressive symptoms varies by sociodemographic and clinical factors. Observational, cross-sectional, cohort study using the Health and Retirement Study. Population-based survey. A total of 3274 individuals who died within 12 months after assessment. Primary outcome: eight-item Center for Epidemiologic Studies Depression Scale (CESD-8). Covariates included sociodemographics, self-reported illnesses, and activity of daily living (ADL) limitations. Average CESD-8 score increased over the last year of life, with 59.3% screening positive for depression in the last month before death. Depression symptoms increased gradually from 12 to 4 months before death (increase of 0.05 points/month; 95% confidence interval [CI] = 0.01-0.08 points/month) and then escalated from 4 to 1 months before death (increase of 0.29 points/month; 95% CI = 0.16-0.39 points/month). Women, younger adults, and nonwhite adults all demonstrated higher rates of depressive symptoms. Individuals with cancer reported escalating rates of depressive symptoms at the end of life, while individuals with lung disease and ADL impairment demonstrated persistently high rates throughout the year before death. This study revealed high rates of depressive symptoms in the last year of life as well as differences in the burden of depressive symptoms. A public health approach must be taken to screen for and appropriately treat symptoms of depression across the lifespan. J Am Geriatr Soc 68:321-328, 2020.


Scientific and ethical concerns in neural fetal tissue transplantation.
This report presents a brief overview of the medical and ethical issues involved with the procurement, preparation, safety, efficacy, and subject protection of human fetal central nervous system tissue in the context of neural transplantation. The ethical perspectives from which to view the clinical use of fetal tissue include the following: 1) that fetal tissue from elective abortions is a surgical specimen; 2) that the use of such tissue involves fetal experimentation in which the fetus is a subject; and 3) that fetal tissue is considered as a cadaveric organ specimen, similar to other organs, but with special considerations compared with adult cadaveric tissue. The latter approach appears to be the most applicable and is parallel to the use of cadaveric organs and tissues after a declaration of brain death. Additional issues include the following: 1) the safety and quality of fetal tissue for implantation; 2) the hypothesis that "legitimization" and "redemption" (potentially positive effects of tissue donation in general) may lead to an increase in elective abortion rates; 3) the ethical issues of the validity and value of human experimentation involving neural grafting; and 4) the type of consent to be obtained and the appropriate timing. Elective abortions, however, probably will continue to be the primary source of fetal tissue for grafting for some time, until other tissue sources become available.


Re-activated adult epicardial progenitor cells are a heterogeneous population molecularly distinct from their embryonic counterparts.
Cardiovascular disease remains the major cause of mortality, and cardiac cell therapy has recently emerged as a paradigm for heart repair. The epicardium is a layer of mesothelial cells covering the heart that during development contributes to different cardiovascular lineages, including cardiomyocytes, but which becomes quiescent after birth. We previously revealed that the peptide thymosin beta 4 (Tβ4) can reactivate adult epicardium-derived cells (EPDCs) after myocardial infarction (MI), to proliferate, and differentiate into cardiovascular derivatives. The aim of this study was to provide a lineage characterization of the adult EPDCs relative to the embryonic epicardial lineage and to determine prospective cell fate biases within the activated adult population during cardiovascular repair. Wt1(GFPCre/+) mice were primed with Tβ4 and MI induced by ligation of the left anterior descending coronary artery. Adult WT1(+) GFP(+) EPDCs were fluorescence-activated cell sorted (FACS) at 2, 4, and 7 days after MI. Embryonic WT1(+) GFP(+) EPDCs were isolated from embryonic hearts (E12.5) by FACS, and sorted cells were characterized by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunostaining. Adult WT1(+) GFP(+) EPDCs were highly heterogeneous, expressing cardiac progenitor and mesenchymal stem markers. Based on the expression of stem cell antigen-1 (Sca-1), CD44, and CD90, we identified different subpopulations of EPDCs of varying cardiovascular potential, according to marker gene profiles, with a molecular phenotype distinct from the source embryonic epicardial cells at E12.5. Thus, adult WT1(+) GFP(+) cells are a heterogeneous population that when activated can restore an embryonic gene programme, but do not revert entirely to adopt an embryonic phenotype. Potential biases in cardiovascular cell fate suggest that discrete subpopulations of EPDCs might be clinically relevant for regenerative therapy.


A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains.
Entry of human immunodeficiency virus type 1 (HIV-1) into cells requires binding to CD4 and fusion with a cellular membrane. Fusion does not occur in most nonhuman cells even when they express human CD4, indicating that one or more human accessory factors are required for virus infection. Recently, a seven-transmembrane domain protein has been shown to serve as an accessory factor for T-cell-tropic (T-tropic) HIV-1 isolates (Y. Feng, C. C. Broder, P. E. Kennedy, and E. A. Berger, Science 272:872-877, 1996). Here we show that expression of this glycoprotein, termed fusin, in murine, feline, simian, and quail cell lines, in conjunction with human CD4, rendered these cells fully permissive for HIV-1 envelope glycoprotein (Env)-mediated membrane fusion. Expression of CD4 or fusin alone did not permit fusion. In addition, introduction of fusin and CD4 into a human cell line, U87MG, that is resistant to HIV-1 induced syncytium formation and to infection by HIV-1 when expressing CD4 alone made this cell line permissive for Env-mediated cell-cell fusion. Fusion was observed only with T-tropic Env proteins. Macrophage-tropic (M-tropic) Env proteins from the SF162, ADA, and Ba-L HIV-1 strains did not fuse with cells expressing fusin and CD4, suggesting that M-tropic viruses utilize an accessory molecule other than fusin. Finally, coexpression of fusin and CD4 made both a murine and feline cell line susceptible to virus infection by T-tropic, but not M-tropic, HIV-1 strains.


Cancer in Women over 50 Years of Age: A Focus on Smoking.
The increase in life expectancy worldwide has resulted in a greater prevalence of chronic non-communicable diseases. This study aims to evaluate the prevalence and factors associated with the occurrence of cancer among Brazilian women over the age of 50. A cross-sectional study with 622 women over the age of 50 was performed using a population survey. The outcome variable was the occurrence of a malignant tumor in any location. The independent variables were sociodemographic characteristics, self-perception of health, health-related habits and morbidities. Statistical analysis was carried out using the chi-square test and Poisson regression. The mean age of the women was 64.1 years. The prevalence of cancer was 6.8%. The main sites of occurrence of malignant tumors were the breast (31.9%), colorectal (12.7%) and skin (12.7%). In the final statistical model, the only factor associated with cancer was smoking > 15 cigarettes/day either currently or in the past: PR 2.03 (95% CI 1.06-3.89). The results have improved understanding of the prevalence and factors associated with cancer in Brazilian women aged 50 years or more. They should be encouraged to maintain a healthy lifestyle and pay particular attention to modifiable risk factors such as smoking.


The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli.
Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity. Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria. However, many design uncertainties remain, including the difficult question of target sequence selection. In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase. The results from this scan pointed to the start codon region as most sensitive to inhibition. To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene. For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective. These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism. Therefore, although other design parameters are also important, the start codon region in E. coli mRNA is the most reliable target site for antisense PNAs.


Use of Community Assessments for Public Health Emergency Response (CASPERs) to Rapidly Assess Public Health Issues - United States, 2003-2012.
Community Assessment for Public Health Emergency Response (CASPER) is an epidemiologic technique designed to provide quick, inexpensive, accurate, and reliable household-based public health information about a community's emergency response needs. The Health Studies Branch at the Centers for Disease Control and Prevention (CDC) provides in-field assistance and technical support to state, local, tribal, and territorial (SLTT) health departments in conducting CASPERs during a disaster response and in non-emergency settings. Data from CASPERs conducted from 2003 through 2012 were reviewed to describe uses of CASPER, ascertain strengths of the CASPER methodology, and highlight significant findings. Through an assessment of the CDC's CASPER metadatabase, all CASPERs that involved CDC support performed in US states and territories from 2003 through 2012 were reviewed and compared descriptively for differences in geographic distribution, sampling methodology, mapping tool, assessment settings, and result and action taken by decision makers. For the study period, 53 CASPERs were conducted in 13 states and one US territory. Among the 53 CASPERS, 38 (71.6%) used the traditional 2-stage cluster sampling methodology, 10 (18.8%) used a 3-stage cluster sampling, and two (3.7%) used a simple random sampling methodology. Among the CASPERs, 37 (69.9%) were conducted in response to specific natural or human-induced disasters, including 14 (37.8%) for hurricanes. The remaining 16 (30.1%) CASPERS were conducted in non-disaster settings to assess household preparedness levels or potential effects of a proposed plan or program. The most common recommendations resulting from a disaster-related CASPER were to educate the community on available resources (27; 72.9%) and provide services (18; 48.6%) such as debris removals and refills of medications. In preparedness CASPERs, the most common recommendations were to educate the community in disaster preparedness (5; 31.2%) and to revise or improve preparedness plans (5; 31.2%). Twenty-five (47.1%) CASPERs documented on the report or publications the public health action has taken based on the result or recommendations. Findings from 27 (50.9%) of the CASPERs conducted with CDC assistance were published in peer-reviewed journals or elsewhere. The number of CASPERs conducted with CDC assistance has increased and diversified over the past decade. The CASPERs' results and recommendations supported the public health decisions that benefitted the community. Overall, the findings suggest that the CASPER is a useful tool for collecting household-level disaster preparedness and response data and generating information to support public health action.


Morphology of the caudal fossa in Cavalier King Charles Spaniels.
Chiari malformations and syringohydromyelia are an important disease complex in Cavalier King Charles Spaniels. Although abnormalities in caudal fossa morphology are considered major contributors to the development of this disease, limited information exists on the range of morphologies in Cavalier King Charles Spaniels and on the relationship of these to clinically evident disease. Sixty-four Cavalier King Charles Spaniels were studied. Each underwent a neurologic examination and magnetic resonance imaging of the cervical spine and brain. T2-weighted sagittal images were used to determine both the morphologic characteristics and volume of the caudal fossa in each dog. This volume was also analyzed as a percentage of total cranial cavity volume. Each attribute was correlated with neurological grade and presence of syringohydromyelia. Fifteen dogs had neurologic signs, and 59 had morphologic abnormalities of the craniocervical junction. While 27 dogs had syringohydromyelia, 13 of these were clinically normal. Cerebellar herniation and occipital dysplasia were common findings but were not associated with syringohydromyelia. Dorsal compressive lesions were noted at the first and second cervical vertebral junction. Factors associated with the presence of neurologic signs included syringohydromyelia and the ratio of caudal fossa/total cranial cavity volume; dogs with signs had significantly larger syringohydromyelia than asymptomatic dogs. Caudal fossa size was not associated with syringohydromyelia. A positive association was identified between foramen magnum size and length of cerebellar herniation. The prevalence of craniocervical junction abnormalities is high in Cavalier King Charles Spaniels. While several factors are associated with neurologic signs, occipital hypoplasia appears to be the most important factor.


Hypomethylating agent 5-aza-2'-deoxycytidine (DAC) ameliorates multiple sclerosis in mouse models.
Increasing evidence supports the role of epigenetics in the development of autoimmune disorders and the possibility of using epigenetic modifying drugs in the context of MS has not yet been investigated. We have explored the effect of the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) in two murine models of experimental allergic encephalomyelitis (EAE). DAC treatment was associated with a significant amelioration of the clinical and histological hallmarks of EAE in both models. These effects were observed both in prophylactic and therapeutic regimens. The milder course of the disease was associated with a reduction in the number of spinal cord infiltrating lymphocytes and amelioration of the histopathological signs associated with EAE. In addition, increased transcript levels of anti-inflammatory cytokines and decreased mRNA expression of pro-inflammatory mediators were also observed. Finally, DAC treatment increased the percentage of circulating regulatory T cells by inducing Foxp3 expression via demethylation of a CpG island in Foxp3.


Increased ventral striatal BOLD activity during non-drug reward anticipation in cannabis users.
Despite an increased understanding of the pharmacology and long-term cognitive effects of cannabis in humans, there has been no research to date examining its chronic effects upon reward processing in the brain. Motivational theories regarding long-term drug use posit contrasting predictions with respect to how drug users are likely to process non-drug incentives. The reward deficiency syndrome (RDS) of addiction posits that there are deficits in dopamine (DA) motivational circuitry for non-drug rewards, such that only drugs of abuse are capable of normalizing DA in the ventral striatum (VS). Alternatively, the opponent process theory (OPT) holds that in individuals prone to drug use, there exists some form of mesolimbic hyperactivity, in which there is a bias towards reward-centred behaviour concomitant with impulsivity. The current study examined BOLD responses during reward and loss anticipation and their outcome deliveries in 14 chronic cannabis users and 14 drug-naive controls during a monetary incentive delay (MID) task. Despite no significant behavioural differences between the two groups, cannabis users had significantly more right VS BOLD activity during reward anticipation. Correlation analyses demonstrated that this right VS BOLD response was significantly correlated with life-time use and reported life-time cannabis joints consumed. No correlations between cannabis abstinence and BOLD responses were observed. We also observed a number of group differences following outcome deliveries, most notably hypoactivity in the left insula cortex in response to loss and loss avoidance outcome notifications in the cannabis group. These results may suggest hypersensitivity during instrumental response anticipation for non-drug rewards and a hyposensitivity to loss outcomes in chronic cannabis users; the implications of which are discussed with respect to the potentially sensitizing effects of cannabis for other rewards.


A model of the pre-assessment learning effects of summative assessment in medical education.
It has become axiomatic that assessment impacts powerfully on student learning. However, surprisingly little research has been published emanating from authentic higher education settings about the nature and mechanism of the pre-assessment learning effects of summative assessment. Less still emanates from health sciences education settings. This study explored the pre-assessment learning effects of summative assessment in theoretical modules by exploring the variables at play in a multifaceted assessment system and the relationships between them. Using a grounded theory strategy, in-depth interviews were conducted with individual medical students and analyzed qualitatively. Respondents' learning was influenced by task demands and system design. Assessment impacted on respondents' cognitive processing activities and metacognitive regulation activities. Individually, our findings confirm findings from other studies in disparate non-medical settings and identify some new factors at play in this setting. Taken together, findings from this study provide, for the first time, some insight into how a whole assessment system influences student learning over time in a medical education setting. The findings from this authentic and complex setting paint a nuanced picture of how intricate and multifaceted interactions between various factors in an assessment system interact to influence student learning. A model linking the sources, mechanism and consequences of the pre-assessment learning effects of summative assessment is proposed that could help enhance the use of summative assessment as a tool to augment learning.


Serum Free Light Chain Assay and κ/λ Ratio Performance in Patients Without Monoclonal Gammopathies: High False-Positive Rate.
Serum free light chain assay is a recommended screening test for monoclonal gammopathies. Anecdotal observations indicated a high rate of false-positive abnormal κ/λ ratios. This study was undertaken to ascertain the magnitude of the false-positive rate and factors contributing to the error rate. Results of serum protein electrophoresis, serum free light chains, and related tests, usually done for investigation of suspected monoclonal gammopathy, were reviewed retrospectively for 270 patients and 297 observations. Using the conventional κ/λ ratio, 36.4% of the ratios were abnormal, in the absence of monoclonal gammopathy. When the renal κ/λ ratio was used, the rate of abnormal κ/λ ratios was 30.1%. In patients with a γ-globulin concentration of 1.6 g/dL or more, the usual κ/λ ratio was abnormal in 54.8% of the patients. Urine protein electrophoresis was used in 53 (19.6%) instances, whereas bone marrow examination was done in 65 (24.1%) cases. Usual κ/λ ratio was abnormal in 36.4% of the observations in patients without evidence of monoclonal gammopathy, and an abnormal κ/λ ratio should not be used as the sole indicator for diagnosis of neoplastic proliferation of the lympho-plasmacytic system. Hypergammaglobulinemia is associated with a higher rate of false-positive abnormal κ/λ ratios. Examination of urine for monoclonal immunoglobulins may be underused, and recommendations by some to use serum free light chain assay in place of, rather than as an adjunct to, urine electrophoresis are not warranted.


Beyond statistical significance: implications of network structure on neuronal activity.
It is a common and good practice in experimental sciences to assess the statistical significance of measured outcomes. For this, the probability of obtaining the actual results is estimated under the assumption of an appropriately chosen null-hypothesis. If this probability is smaller than some threshold, the results are deemed statistically significant and the researchers are content in having revealed, within their own experimental domain, a "surprising" anomaly, possibly indicative of a hitherto hidden fragment of the underlying "ground-truth". What is often neglected, though, is the actual importance of these experimental outcomes for understanding the system under investigation. We illustrate this point by giving practical and intuitive examples from the field of systems neuroscience. Specifically, we use the notion of embeddedness to quantify the impact of a neuron's activity on its downstream neurons in the network. We show that the network response strongly depends on the embeddedness of stimulated neurons and that embeddedness is a key determinant of the importance of neuronal activity on local and downstream processing. We extrapolate these results to other fields in which networks are used as a theoretical framework.


Evaluation of systemic hemodynamic effects on post-transplant perfusion to renal allograft.
Hemodynamic parameters and post-transplant renal perfusion were evaluated in 24 cadaver kidney transplantation. Cardiac output (CO), systemic vascular resistance (SVR) and mean arterial pressure (MAP), were continuously monitored in each patient via a transesophageal Doppler cardiac output monitor and radial arterial catheter. Seventeen were well perfused (Group 1) after releasing vascular clamps. Six among group 1 presented as the clinical picture of acute tubular necrosis. Seven were poorly perfused (Group 2). Post-anastomotic CO significantly increased in group 1, and decreased in group 2. SVR significantly decreased in group 1, and increased in group 2. MAP were not significantly different between group 1 and 2. Treatments based on hemodynamic derangement were initiated as soon as it was detected. Correction of CO was associated with the improvement of post-anastomotic renal perfusion, while SVR and MAP were not. Among the 7 poorly perfused kidneys, 5 became well perfused before leaving the operating room. The remaining 2 resumed their hemodynamic stability and renal function a few days later after oral captopril was administered. Two among the 6 acute tubular necrosis resumed renal function as long as hemodynamic stability was restored. Our study demonstrates that hemodynamic parameters are parallel to renal perfusion status. Restoration of hemodynamic derangement not only improves renal perfusion but also renal function. It is concluded that hemodynamic instability and graft ischemic time contribute to the variation of post-transplant perfusion to renal allograft. Non-invasive cardiac output monitor is a safe and reliable monitor which we recommend to be used during the operation of kidney transplantation.


Leukoencephalopathy with thalamus and brainstem involvement and high lactate 'LTBL' caused by EARS2 mutations.
In the large group of genetically undetermined infantile-onset mitochondrial encephalopathies, multiple defects of mitochondrial DNA-related respiratory-chain complexes constitute a frequent biochemical signature. In order to identify responsible genes, we used exome-next-generation sequencing in a selected cohort of patients with this biochemical signature. In an isolated patient, we found two mutant alleles for EARS2, the gene encoding mitochondrial glutamyl-tRNA synthetase. The brain magnetic resonance imaging of this patient was hallmarked by extensive symmetrical cerebral white matter abnormalities sparing the periventricular rim and symmetrical signal abnormalities of the thalami, midbrain, pons, medulla oblongata and cerebellar white matter. Proton magnetic resonance spectroscopy showed increased lactate. We matched this magnetic resonance imaging pattern with that of a cohort of 11 previously selected unrelated cases. We found mutations in the EARS2 gene in all. Subsequent detailed clinical and magnetic resonance imaging based phenotyping revealed two distinct groups: mild and severe. All 12 patients shared an infantile onset and rapidly progressive disease with severe magnetic resonance imaging abnormalities and increased lactate in body fluids and proton magnetic resonance spectroscopy. Patients in the 'mild' group partially recovered and regained milestones in the following years with striking magnetic resonance imaging improvement and declining lactate levels, whereas those of the 'severe' group were characterized by clinical stagnation, brain atrophy on magnetic resonance imaging and persistent lactate increases. This new neurological disease, early-onset leukoencephalopathy with thalamus and brainstem involvement and high lactate, is hallmarked by unique magnetic resonance imaging features, defined by a peculiar biphasic clinical course and caused by mutations in a single gene, EARS2, expanding the list of medically relevant defects of mitochondrial DNA translation.


Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level.
Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE formation. Final DE differentiation was also analyzed with immunocytochemistry and single-cell gene expression profiling. We found that cells treated with activin A in combination with sodium butyrate and B27 serum-free supplement medium generated the most mature DE cells. Cell population studies were useful to monitor the temporal expression of genes involved in primitive streak formation and endoderm formation, while single-cell analysis allowed us to study cell culture heterogeneity and fingerprint individual cells. In addition, single-cell analysis revealed distinct gene expression patterns for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize cell types and subpopulations.


The Impact of Diabetes on Breast Cancer Treatments and Outcomes: A Population-Based Study.
Women with breast cancer and diabetes face worse outcomes than those with breast cancer without diabetes; however, the contribution of comorbidity to these disparities remains unclear. We evaluated the impact of diabetes on receipt of cancer treatments as well as mortality while accounting for other comorbidities. Ontario administrative databases were used to compare the rate of receipt of breast cancer treatments between women with and without diabetes. We also performed adjusted cause-specific hazard models to account for comorbidities when evaluating differences in treatments received and mortality outcomes between the two groups. Women with diabetes and stage III breast cancer were slightly less likely to receive chemotherapy (relative risk [RR] 0.93 [95% CI 0.89-0.97]), although this difference was not significant when we adjusted for comorbidities (adjusted hazard ratio [aHR] 1.03 [95% CI 0.93-1.13]). We saw similar trends for receipt of guideline-adherent radiotherapy (RR 0.97 [0.95-0.99], aHR 0.98 [0.94-1.02]). All-cause mortality was increased in women with diabetes after adjusting for comorbidities (aHR 1.16 [1.06-1.27]), but breast cancer-specific mortality was not increased overall. Women with a longer duration of diabetes and those with preexisting cardiovascular disease had increased all-cause and cancer-specific mortality. Although cancer treatments received were similar between women with and without diabetes, breast cancer-specific mortality remains higher among women with diabetes who have longer diabetes duration or preexisting cardiovascular disease. This study uncovers new information about key risk factors for poorer prognosis in women with diabetes and breast cancer.


Virulence mechanisms displayed by Salmonella to impair dendritic cell function.
Dendritic cells (DCs) link innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) on bacteria. DCs can capture and degrade bacteria and present their antigens on MHC molecules to T cells. PAMP recognition promotes DC maturation, a phenotypic change that empowers them to prime naïve T cells. As a result, an adaptive immune response that specifically targets bacteria-derived antigens is initiated. Consequently, any impairment of DC function might contribute to bacterial survival and dissemination in the host. Therefore, the characterization of DC-bacteria interactions is required to understand the mechanisms used by virulent bacteria to avoid adaptive immunity. An example of a bacterial pathogen capable of interfering with DC function is Salmonella enterica serovar Typhimurium (S. Typhimurium), which causes a typhoid-like disease in mice. Virulent strains of S. Typhimurium are able to differentially modulate the entrance to DCs and avoid lysosomal degradation, to prevent antigen presentation on MHC molecules. These features of virulent S. Typhimurium are controlled by virulence factors encoded by Salmonella Pathogenicity Islands 1 and 2. Modulation of DC functions by the activity of these gene products is supported by several recent studies, which have shown that pathogenesis might depend on this attribute of virulent S. Typhimurium. Here we discuss recent data showing that several virulence factors from Salmonella are required to differentially modulate DC function and adaptive immunity in the host.


Reliability and validity of non-radiographic methods of thoracic kyphosis measurement: a systematic review.
A wide array of instruments are available for non-invasive thoracic kyphosis measurement. Guidelines for selecting outcome measures for use in clinical and research practice recommend that properties such as validity and reliability are considered. This systematic review reports on the reliability and validity of non-invasive methods for measuring thoracic kyphosis. A systematic search of 11 electronic databases located studies assessing reliability and/or validity of non-invasive thoracic kyphosis measurement techniques. Two independent reviewers used a critical appraisal tool to assess the quality of retrieved studies. Data was extracted by the primary reviewer. The results were synthesized qualitatively using a level of evidence approach. 27 studies satisfied the eligibility criteria and were included in the review. The reliability, validity and both reliability and validity were investigated by sixteen, two and nine studies respectively. 17/27 studies were deemed to be of high quality. In total, 15 methods of thoracic kyphosis were evaluated in retrieved studies. All investigated methods showed high (ICC ≥ .7) to very high (ICC ≥ .9) levels of reliability. The validity of the methods ranged from low to very high. The strongest levels of evidence for reliability exists in support of the Debrunner kyphometer, Spinal Mouse and Flexicurve index, and for validity supports the arcometer and Flexicurve index. Further reliability and validity studies are required to strengthen the level of evidence for the remaining methods of measurement. This should be addressed by future research.


Fosfomycin in infections caused by multidrug-resistant Gram-negative pathogens.
The alarming increase in antibiotic resistance rates reported for various pathogens has resulted in the use of alternative treatment policies. Given the fairly limited availability of new antimicrobial drugs, the reassessment of older antibiotics is now an interesting option. Fosfomycin, a bactericidal analog of phosphoenolpyruvate that has been previously been employed as an oral treatment for uncomplicated urinary tract infection, has recently raised interest among physicians worldwide. In general, the advanced resistance described in Gram-negative bacteria suggests that fosfomycin can be an appropriate treatment option for patients with highly resistant microbial infections. This review, which refers to key available data, focuses on the possibility of extending the use of fosfomycin beyond urinary tract infections and against multidrug-resistant Gram-negative bacteria.


Control-independent mosaic single nucleotide variant detection with DeepMosaic.
Mosaic variants (MVs) reflect mutagenic processes during embryonic development and environmental exposure, accumulate with aging and underlie diseases such as cancer and autism. The detection of noncancer MVs has been computationally challenging due to the sparse representation of nonclonally expanded MVs. Here we present DeepMosaic, combining an image-based visualization module for single nucleotide MVs and a convolutional neural network-based classification module for control-independent MV detection. DeepMosaic was trained on 180,000 simulated or experimentally assessed MVs, and was benchmarked on 619,740 simulated MVs and 530 independent biologically tested MVs from 16 genomes and 181 exomes. DeepMosaic achieved higher accuracy compared with existing methods on biological data, with a sensitivity of 0.78, specificity of 0.83 and positive predictive value of 0.96 on noncancer whole-genome sequencing data, as well as doubling the validation rate over previous best-practice methods on noncancer whole-exome sequencing data (0.43 versus 0.18). DeepMosaic represents an accurate MV classifier for noncancer samples that can be implemented as an alternative or complement to existing methods.


Protease-resistant prion protein produced in vitro lacks detectable infectivity.
The 'protein-only' hypothesis of prion propagation argues that infectious prions consist of PrP(Sc), a conformational isomer of host-derived prion protein (PrP(C)), which can be distinguished from PrP(C) by its partial resistance to proteases. While protease-resistant PrP has been produced by mixing PrP(Sc) and recombinant-derived PrP(C) in vitro, bioassay of any new infectivity has been precluded by the need to use a large molar excess of same species PrP(Sc). Transgenic mice expressing a chimaeric hamster-mouse PrPC (MH2M PrP(C)) are, unlike conventional mice, highly susceptible to Sc237 hamster scrapie. In addition, they produce MH2M PrP(Sc) and infectivity which is pathogenic for conventional mice. We have therefore attempted to produce MH2M PrP(Sc) in vitro as any infectivity produced could be distinguished from the hamster PrP(Sc) used to promote the conversion by bioassay in conventional mice. Although protease-resistant MH2M PrP was produced, no infectivity was detected on bioassay. These results argue that acquisition of protease resistance by PrP(C) is not sufficient for the propagation of infectivity.


Public counselling: Brazilian Pentecostal intimate performances among urban women in Mozambique.
Analysing the public character of Brazilian Pentecostal counselling sessions in urban Mozambique, this paper aims to examine the configurations of exposure through which counselling acquires and produces sociocultural forms of intimacy. During 'therapy of love' sessions held by the Brazilian Universal Church of the Kingdom of God, strict bodily acts need to be performed publicly to authorise love. Drawing on the notion of religious habitus, the paper focuses on how the performativity of Brazilian Pentecostal counselling practices responds to and shapes a public culture of love and intimacy among upwardly mobile women in urban Mozambique, and how the performative scripts converts carry out during therapy result in ambiguous relationships.


Swim Positioning and its Influence on Triathlon Outcome.
Questions have been raised regarding which of the three legs of a triathlon influences the final finishing position. Some coaches subjectively believe that the swim and run are more important than the cycle, especially since the introduction of drafting during the cycle. This study analysed race position shifts between each of the three disciplines to assess the importance of the swim finish position and final finish position during draft legal Olympic distance triathlon events. Ten male and 10 female triathlon world cup events during one season were analysed. The results suggested that the triathlon swim leg is important because the winner exited the water in the first pack in 90% of elite male and 70% of elite female races. Correlations were also derived from finishing order for the whole triathlon and a finishing order that included the swim only, cycle only or run only time. For men, the average correlations for final finishing order with each of the swim, cycle and run, respectively, were 0.49, 0.67 and 0.86 and for the women; average correlations were 0.39, 0.67 and 0.85. Hence, this indicated that it was important to exit the water in the first pack and run well after cycling to achieve a successful final finishing position.


Neuroprotective effects of a novel carnosine-hydrazide derivative on hippocampal CA1 damage after transient cerebral ischemia.
Ischemia-reperfusion injuries produce reactive oxygen species that promote the peroxide lipid oxidation process resulting in the production of an endogenic lipid peroxide, 4-hydroxy-trans-2-nonenal (4-HNE), a highly cytotoxic aldehyde that induces cell death. We synthesized a novel 4-HNE scavenger - a carnosine-hydrazide derivative, l-carnosine hydrazide (CNN) - and examined its neuroprotective effect in a model of transient ischemia. PC-12 cells were pre-incubated with various doses (0-50 mmol/L) of CNN for 30 min, followed by incubation with 4-HNE (250 μM). An MTT assay was performed 24 h later to examine cell survival. Transient ischemia was induced by bilateral common carotid artery occlusion (BCCO) in the Mongolian gerbil. Animals were assigned to sham-operated (n = 6), placebo-treated (n = 12), CNN pre-treated (20 mg/kg; n = 12), CNN post-treated (100 mg/kg; n = 11), and histidyl hydrazide (a previously known 4-HNE scavenger) post-treated (100 mg/kg; n = 7) groups. Heat shock protein 70 immunoreactivity in the hippocampal CA1 region was evaluated 24 h later, while delayed neuronal death using 4-HNE staining was evaluated 7 days later. Pre-incubation with 30 mmol/L CNN completely inhibited 4-HNE-induced cell toxicity. CNN prevented delayed neuronal death by >60% in the pre-treated group (p < 0.001) and by >40% in the post-treated group (p < 0.01). Histidyl hydrazide post-treatment elicited no protective effect. CNN pre-treatment resulted in high heat shock protein 70 and low 4-HNE immunoreactivity in CA1 pyramidal neurons. Higher 4-HNE immunoreactivity was also found in the placebo-treated animals than in the CNN pre-treated animals. Our novel compound, CNN, elicited highly effective 4-HNE scavenging activity in vitro. Furthermore, CNN administration both pre- and post-BCCO remarkably reduced delayed neuronal death in the hippocampal CA1 region via its induction of heat shock protein 70 and scavenging of 4-HNE.


What ever happened to N-of-1 trials? Insiders' perspectives and a look to the future.
When feasible, randomized, blinded single-patient (n-of-1) trials are uniquely capable of establishing the best treatment in an individual patient. Despite early enthusiasm, by the turn of the twenty-first century, few academic centers were conducting n-of-1 trials on a regular basis. The authors reviewed the literature and conducted in-depth telephone interviews with leaders in the n-of-1 trial movement. N-of-1 trials can improve care by increasing therapeutic precision. However, they have not been widely adopted, in part because physicians do not sufficiently value the reduction in uncertainty they yield weighed against the inconvenience they impose. Limited evidence suggests that patients may be receptive to n-of-1 trials once they understand the benefits. N-of-1 trials offer a unique opportunity to individualize clinical care and enrich clinical research. While ongoing changes in drug discovery, manufacture, and marketing may ultimately spur pharmaceutical makers and health care payers to support n-of-1 trials, at present the most promising resuscitation strategy is stripping n-of-1 trials to their essentials and marketing them directly to patients. In order to optimize statistical inference from these trials, empirical Bayes methods can be used to combine individual patient data with aggregate data from comparable patients.


Trabecular meshwork bradykinin receptors: mRNA levels, immunohistochemical visualization, signaling processes pharmacology, and linkage to IOP reduction.
To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. Previously documented procedures were utilized throughout these studies. Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-β2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 μg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 μg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.


Understanding context dependency in the response of forest understorey plant communities to nitrogen deposition.
Understorey communities can dominate forest plant diversity and strongly affect forest ecosystem structure and function. Understoreys often respond sensitively but inconsistently to drivers of ecological change, including nitrogen (N) deposition. Nitrogen deposition effects, reflected in the concept of critical loads, vary greatly not only among species and guilds, but also among forest types. Here, we characterize such context dependency as driven by differences in the amounts and forms of deposited N, cumulative deposition, the filtering of N by overstoreys, and available plant species pools. Nitrogen effects on understorey trajectories can also vary due to differences in surrounding landscape conditions; ambient browsing pressure; soils and geology; other environmental factors controlling plant growth; and, historical and current disturbance/management regimes. The number of these factors and their potentially complex interactions complicate our efforts to make simple predictions about how N deposition affects forest understoreys. We review the literature to examine evidence for context dependency in N deposition effects on forest understoreys. We also use data from 1814 European temperate forest plots to test the ability of multi-level models to characterize context-dependent understorey responses across sites that differ in levels of N deposition, community composition, local conditions and management history. This analysis demonstrated that historical management, and plot location on light and pH-fertility gradients, significantly affect how understorey communities respond to N deposition. We conclude that species' and communities' responses to N deposition, and thus the determination of critical loads, vary greatly depending on environmental contexts. This complicates our efforts to predict how N deposition will affect forest understoreys and thus how best to conserve and restore understorey biodiversity. To reduce uncertainty and incorporate context dependency in critical load setting, we should assemble data on underlying environmental conditions, conduct globally distributed field experiments, and analyse a wider range of habitat types.


IL-13 selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells.
Previous studies with human umbilical vein endothelial cells (HUVEC) have shown that the cytokine IL-4 induces adherence of human eosinophils, but not neutrophils, because of its ability to selectively induce surface expression of vascular cell adhesion molecule-1 (VCAM-1). Because the cytokine IL-13 shares a number of biologic properties with IL-4, we examined the effect of IL-13 on the expression and function of adhesion molecules on HUVEC. Incubation of HUVEC for 4 to 48 h with IL-13 (0.1 to 15 U/ml) induced surface expression of VCAM-1, as detected by indirect immunofluorescence and flow cytometry, without significantly affecting expression of E-selectin or intercellular adhesion molecule-1. The kinetics and maximal IL-13-induced expression of VCAM-1 were similar to those seen with IL-4. Treatment of HUVEC with an optimal concentration of IL-13 (15 U/ml for 24 h) induced adhesiveness for eosinophils, but not for neutrophils, and adhesion was completely inhibited by mAb recognizing VCAM-1 or alpha 4 integrin (CD49d). These results demonstrate that IL-13, like IL-4, selectively stimulates HUVEC to express functional cell surface VCAM-1 and suggest a possible role for IL-13 in promoting VCAM-1/alpha 4 integrin-dependent accumulation of eosinophils during allergic and other inflammatory reactions in vivo.


Comparative mass spectrometric and immunoassay-based proteome analysis in serum of Duchenne muscular dystrophy patients.
Duchenne muscular dystrophy (DMD) is a severe and fatal neuromuscular disease. With the current developments on novel therapeutic strategies for DMD, the need to carefully monitor disease progression or regression upon treatment using molecular markers has become urgent. 2D LC protein fractionation was performed on patient serum samples, followed by LC-MS/MS-based identifications with label-free quantifications. Protein signatures were compared between patients and healthy (child and adult) controls and between ambulant and nonambulant patients. Various myofibrillar proteins demonstrated differences between DMD patients and controls, likely due to leakiness and breakdown of muscle fibers. Previously reported biomarkers, such as muscle-derived titin, myosin, and carbonic anhydrase I (CA1), were verified. MS-based results were compared with ELISA for vitamin D binding protein (GC), fibulin-1 (FBLN1), gelsolin (GSN), and carbonic anhydrase 1 (CA1). The combined results of MS- and ELISA-based quantifications indicated more studies are needed to validate this serum protein signature for DMD patients. With these data promising candidate biomarkers have been identified for a rare genetic disease using serum proteome analysis.


Anaemia in adolescent girls: An intervention of diet editing and counselling.
Though a major public health problem of nutritional anaemia in schoolchildren is being addressed by iron supplementation and/or fortified food, they continue to be anaemic. We aimed to study the effect of fluoride consumption on haemoglobin levels and whether elimination of fluoride from the diet would correct anaemia in children. Two hundred and fifty adolescent girls, 10-17 years of age, from a government senior secondary girls school in East Delhi, participated in the programme. Only those girls who were dewormed in the school health programme and not on any medication particularly for malaria, were included. The investigations done were (i) haemoglobin level; (ii) fluoride content in urine; and (iii) fluoride content in drinking water both at home and in school. The anaemic students consuming safe drinking water with fluoride level <1.0 mg/L and with urine fluoride >1.0 mg/L were introduced to interventions, viz. diet editing and diet counselling when parents came for the monthly parent-teacher meeting. Besides the parents, their wards and class teachers also attended the counselling session. The students were monitored by re-testing haemoglobin and urine fluoride levels at 1, 3 and 6 months after the start of the intervention. There was an inverse relationship in the levels of urine fluoride and haemoglobin. Reduction in the level of urine fluoride led to a rise in the haemoglobin level. Following interventions, the haemoglobin level revealed significant improvement from the anaemic (<12.0 g/dl) to the non- anaemic range (12.0-14.4 g/dl). At 6 months of follow-up, of the 244 girls studied, those with severe anaemia decreased from 3% to 1%, with moderate anaemia from 97% to58% and the non-anaemic girls increased from 0% to 41%. Non-toxic nutritive food and safe water with fluoride level < 1.0 mg/L are useful in improving haemoglobin levels in a high percentage of anaemic schoolchildren. A haemoglobin level of > 12.0-14.4 g/dL is an achievable target in children without iron supplementation.


Incidence and treatment of pulmonary vein stenosis after repair of total anomalous pulmonary venous connection.
In this study, we aimed to investigate the incidence and treatment of pulmonary vein stenosis after repair of total anomalous pulmonary venous connection. Between December 2010 and December 2016, a total of 40 patients (25 males, 15 females; mean age 45±41 days; range, 2 days to 6 months) who underwent total anomalous pulmonary venous connection repair were retrospectively analyzed. Eighteen (45%) of the patients were supracardiac, seven (17.5%) were cardiac, eight (20%) were infracardiac, and seven (17.5%) were mixed-type anomaly. Twelve patients (30%) had obstruction of the pulmonary venous pathways. Hospital mortality was seen in three patients (7.5%). All of non-survivors received emergent surgery and the causes of death were sepsis and multiple organ failure. A total of 23 complications were seen in 20 patients (50%) mainly delayed sternal closure (n=11; 27.5%) and prolonged mechanical ventilation (n=6; 15%). The mean follow-up was 12.2±9.6 (range, 1 to 36) months in all patients, except one. Pulmonary vein stenosis developed in three patients (8.3%) after repair. All of them underwent sutureless repair. Total anomalous pulmonary venous connection can be repaired with low mortality and morbidity rates and favorable mid-term outcomes. Close follow-up is necessary due to the risk of development of pulmonary vein stenosis. Sutureless repair may provide relief in such cases.


Inhibition and induction of hepatic drug metabolism in rats and mice by nafimidone and its major metabolite nafimidone alcohol.
Nafimidone, a candidate anticonvulsant agent, and its metabolite (nafimidone alcohol) demonstrated potent inhibition of hepatic drug metabolism in male rats. Inhibition occurred both in vivo following acute administration as a prolongation of hexobarbital sleeping time and as an elevation of plasma hexobarbital levels, and in vitro following addition to hepatic microsomes as a disruption of ethyl-morphine N-demethylation and aniline p-hydroxylation. Inhibition of ethylmorphine N-demethylation was of a mixed type, whereas aniline p-hydroxylation was inhibited in a noncompetitive manner; the micromolar Ki values obtained for both enzymes were severalfold lower than those obtained for imidazole. Nafimidone alcohol produced a type II difference spectrum when added to rat hepatic microsomes. The Ks value was 2.1 microM. Chronic administration of nafimidone alcohol caused induction of hepatic drug metabolism typified by shortening of pentobarbital sleeping time in vivo in male mice and a doubling of hepatic microsomal cytochrome P-450 content in male rats. In rats, these changes were associated with a 30-fold elevation in the Vmax for microsomal ethoxyresorufin O-deethylase and a moderate increase in the Vmax for microsomal ethylmorphine N-demethylase, but no change in either the rate of aniline p-hydroxylation, 4-hydroxybiphenyl- or 4-methylumbelliferone UDP-glucuronosyltransferase, or the activity of the flavoprotein reductase component. These data suggest induction of a predominating cytochrome P-448-type of Phase I drug-metabolizing activity by nafimidone alcohol.


Magnetic domain coupling study in single-crystalline Fe/CoO bilayers.
We report on a study of the magnetic domain coupling in epitaxial wedge-shaped Fe layers deposited onto CoO/Ag(001). By using photoelectron emission microscopy (PEEM) in combination with x-ray magnetic circular and linear dichroism (XMCD, XMLD), we imaged the ferromagnetic and antiferromagnetic domains present in the Fe and CoO layers, respectively, below the CoO magnetic ordering temperature. The uncompensated Co spins at the Fe/CoO interface were revealed by XMCD-PEEM and were found to be coupled parallel to the magnetization of the Fe layer. An increase of the CoO XMLD contrast is visible for Fe thicknesses below 2 ML, where the Fe layer lacks magnetic long-range order.


Addressing the needs of caregivers of cancer patients in general practice: a complex intervention.
This study aimed to develop an innovation to assist general practitioners (GPs) in Australia to proactively address the needs of caregivers of people with cancer. Six GPs were video recorded each consulting six actor-patients in their respective practices. All cases depicted caregivers of people with cancer. The patients were instructed to complete a Needs Assessment Tool for Caregivers (NAT-C), before the consultation. Actor-patients were instructed to present the NAT-C to three of the six GPs they consulted, selected at random. Two assessors independently reviewed each consultation performance using the Leicester Assessment Package (LAP). The practitioners and actor-patients focused on the value of the NAT-C and how it could be deployed to best effect in a subsequent 'stimulated recall session'. Thirty-four consultations were successfully recorded. The mean duration of consultations was 13 min. 47 sec. (range 6 min. 3 sec. to 22 min. 51 sec.). GPs differed in core competencies as measured by the LAP (P<0.001), range 37-92%. However, they demonstrated no significant differences in performance (LAP scores) analysed by scenario (P = 0.99). The 'generalised estimating equation' (GEE) model identified an improved LAP score in consultations in which the NAT-C was used (average of 3.3 points; 95% CI: -3.99, 10.6), after controlling for the different GPs and scenarios, but this improvement was not statistically significant (P = 0.37). The participants felt that the NAT-C was beneficial and suggested how it could be further refined. If this innovation had been formally tested in a randomised trial without assessing its impact on the consultation there might have been significant difficulties with administering the intervention in practice.


Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells.
Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.


Ionic matrix for enhanced MALDI imaging mass spectrometry for identification of phospholipids in mouse liver and cerebellum tissue sections.
The ionic matrix (IM) is considered to be versatile for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the identification of a wide range of biomolecules due to its good solubility for a variety of analytes, formation of homogeneous crystals with analytes, and high vacuum stability. When these advantages are exploited, the performance of IM of α-cyano-4-hydroxycinnamic acid butylamine (CHCAB) and 2,5-dihydroxybenzoic acid butylamine (DHBB) was compared with other matrixes for the identification of phospholipids in standard mixtures and mouse liver tissue sections. The results showed that the IM of CHCAB caused higher signal intensity and allowed the detection of a number phospholipids such as phosphatidylethanolamine (PE) and phosphatidylserine (PS) in addition to detection of phosphatidylcholine (PC) on the surface of the liver tissue sample. The IM of CHCAB was also used to identify the species of lipids present in different layers of cerebellum where the greater numbers of biomolecules were detected as compared to DHB matrix. Further, the feasibility of the proposed method was extended for the analysis of tryptic digested cytochrome c for increased signal intensity and number of peptide sequences in MALDI-MS. Thus, the application of IM to MALDI-MS could be a promising tool for imaging biomolecules in tissue sections in high throughput analyses with high sensitivity.


Effect of metformin vs placebo on and metabolic factors in NCIC CTG MA.32.
Metformin may improve metabolic factors (insulin, glucose, leptin, highly sensitive C-reactive protein [hs-CRP]) associated with poor breast cancer outcomes. The NCIC Clinical Trials Group (NCIC CTG) MA.32 investigates effects of metformin vs placebo on invasive disease-free survival and other outcomes in early breast cancer. Maintaining blinding of investigators to outcomes, we conducted a planned, Data Safety Monitoring Committee-approved, analysis of the effect of metformin vs placebo on weight and metabolic factors at six months, including examination of interactions with baseline body mass index (BMI) and insulin, in the first 492 patients with paired blood samples. Eligible nondiabetic subjects with T1-3, N0-3, M0 breast cancer who had completed surgery and (neo)adjuvant chemotherapy (if given) provided fasting plasma samples at random assignment and at six months. Glucose was measured locally; blood was aliquoted, frozen, and stored at -80°C. Paired plasma aliquots were analyzed for insulin, hs-CRP, and leptin. Spearman correlation coefficients were calculated and comparisons analyzed using Wilcoxon signed rank test. All statistical tests were two-sided. Mean age was 52.1±9.5 years in the metformin group and 52.6 ± 9.8 years in the placebo group. Arms were balanced for estrogen/progesterone receptor, BMI, prior (neo)adjuvant chemotherapy, and stage. At six months, decreases in weight and blood variables were statistically significantly greater in the metformin arm (vs placebo) in univariate analyses: weight -3.0%, glucose -3.8%, insulin -11.1%, homeostasis model assessment -17.1%, leptin -20.2%, hs-CRP -6.7%; all P values were less than or equal to .03. There was no statistically significant interaction of change in these variables with baseline BMI or insulin. Metformin statistically significantly improved weight, insulin, glucose, leptin, and CRP at six months. Effects did not vary by baseline BMI or fasting insulin.


Micrographia of the twenty-first century: from camera obscura to 4D microscopy.
In this paper, the evolutionary and revolutionary developments of microscopic imaging are overviewed with a perspective on origins. From Alhazen's camera obscura, to Hooke and van Leeuwenhoek's two-dimensional optical micrography, and on to three- and four-dimensional (4D) electron microscopy, these developments over a millennium have transformed humans' scope of visualization. The changes in the length and time scales involved are unimaginable, beginning with the visible shadows of candles at the centimetre and second scales, and ending with invisible atoms with space and time dimensions of sub-nanometre and femtosecond. With these advances it has become possible to determine the structures of matter and to observe their elementary dynamics as they unfold in real time. Such observations provide the means for visualizing materials behaviour and biological function, with the aim of understanding emergent phenomena in complex systems.


Right across the tree of life: the evolution of left-right asymmetry in the Bilateria.
Directional left/right (LR) asymmetries, in which there are consistent, heritable differences in morphology between the left and right sides of bilaterally symmetrical organisms, are found in animals across the Bilateria. For many years, we have lacked evidence for shared mechanisms underlying their development. This led to the supposition that the mechanisms driving establishment of LR asymmetries, and consequently the asymmetries themselves, had evolved separately in the three major Superphyla that constitute the Bilateria. The recent discovery that the transforming growth factor-beta (TGF-B) ligand Nodal plays a role in the regulation of LR asymmetry in both Deuterostomia and Lophotrochozoa has reignited debate in this field, as it suggests that at least this aspect of the development of the LR axis is conserved. In this review, we discuss evidence for shared mechanisms of LR asymmetry establishment across the bilaterian tree of life and consider how these mechanisms might have diverged across the Metazoa over the last 500 million years or so of evolution. As well as the likelihood that Nodal is an ancestral mechanism for regulating LR asymmetry, we reemphasize cytoskeletal architecture as a potential shared mechanism underlying symmetry breaking. However, convergent evolution remains a distinct possibility and study of a wider diversity of species will be needed to distinguish between conserved and lineage-specific mechanisms.


Genetic consequences of postglacial range expansion in two codistributed rodents (genus Dipodomys) depend on ecology and genetic locus.
How does range expansion affect genetic diversity in species with different ecologies, and do different types of genetic markers lead to different conclusions? We addressed these questions by assessing the genetic consequences of postglacial range expansion using mitochondrial DNA (mtDNA) and nuclear restriction site-associated DNA (RAD) sequencing in two congeneric and codistributed rodents with different ecological characteristics: the desert kangaroo rat (Dipodomys deserti), a sand specialist, and the Merriam's kangaroo rat (Dipodomys merriami), a substrate generalist. For each species, we compared genetic variation between populations that retained stable distributions throughout glacial periods and those inferred to have expanded since the last glacial maximum. Our results suggest that expanded populations of both species experienced a loss of private mtDNA haplotypes and differentiation among populations, as well as a loss of nuclear single-nucleotide polymorphism (SNP) private alleles and polymorphic loci. However, only D. deserti experienced a loss of nucleotide diversity (both mtDNA and nuclear) and nuclear heterozygosity. For all indices of diversity and differentiation that showed reduced values in the expanded areas, D. deserti populations experienced a greater degree of loss than did D. merriami populations. Additionally, patterns of loss in genetic diversity in expanded populations were substantially less extreme (by two orders of magnitude in some cases) for nuclear SNPs in both species compared to that observed for mitochondrial data. Our results demonstrate that ecological characteristics may play a role in determining genetic variation associated with range expansions, yet mtDNA diversity loss is not necessarily accompanied by a matched magnitude of loss in nuclear diversity.


Controlling hot electrons by wave amplification and decay in compressing plasma.
Through particle-in-cell simulations, it is demonstrated that a part of the mechanical energy of compressing plasma can be controllably transferred to hot electrons by preseeding the plasma with a Langmuir wave that is compressed together with the medium. Initially, a wave is undamped, so it is amplified under compression due to plasmon conservation. Later, as the phase velocity also changes under compression, Landau damping can be induced at a predetermined instant of time. Then the wave energy is transferred to hot electrons, shaping the particle distribution over a controllable velocity interval, which is wider than that in stationary plasma. For multiple excited modes, the transition between the adiabatic amplification and the damping occurs at different moments; thus, individual modes can deposit their energy independently, each at its own prescribed time.


A novel nanozyme assay utilising the catalytic activity of silver nanoparticles and SERRS.
Artificial enzymes have become an increasingly interesting area of research due to their many advantages over natural protein enzymes which are expensive, difficult to isolate and unable to stand harsh environments. An important area of this research involves using metal nanoparticles as artificial enzymes, known as nanozymes, which exhibit peroxidase-like activity enabling them to catalyse the oxidation of substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), giving a colorimetric response. Here we exploit the catalytic activity of silver nanoparticles (Ag NPs) in a surface based silver-linked immunosorbent assay (SLISA) to detect human C-reactive protein (CRP), an inflammatory marker. Ag NPs were conjugated to antibodies with specific recognition for the corresponding target antigenic molecule, CRP, and the Ag NPs were used to catalyse the oxidation of TMB by H2O2. The resulting coloured oxidation product was detected using SERRS. We demonstrate that Ag NPs can replace the enzymes used in a conventional ELISA and a detection limit of 1.09 ng mL-1 of CRP can be achieved. It indicates the promise for SLISAs for biomarker detection and opens the way for further assays of this nature to be created. This novel assay has the potential to be optimised to detect lower levels of CRP and can be further extended for the sensitive and specific detection of other relevant biomarkers.


Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis.
Friedreich's ataxia (FRDA) is a devastating, multisystemic disorder caused by recessive mutations in the mitochondrial protein frataxin (FXN). FXN participates in the biosynthesis of Fe-S clusters and is considered to be essential for viability. Here we report that when grown in 1% ambient O2, FXN null yeast, human cells, and nematodes are fully viable. In human cells, hypoxia restores steady-state levels of Fe-S clusters and normalizes ATF4, NRF2, and IRP2 signaling events associated with FRDA. Cellular studies and in vitro reconstitution indicate that hypoxia acts through HIF-independent mechanisms that increase bioavailable iron as well as directly activate Fe-S synthesis. In a mouse model of FRDA, breathing 11% O2 attenuates the progression of ataxia, whereas breathing 55% O2 hastens it. Our work identifies oxygen as a key environmental variable in the pathogenesis associated with FXN depletion, with important mechanistic and therapeutic implications.


The flavonoid pathway in tomato seedlings: transcript abundance and the modeling of metabolite dynamics.
Flavonoids are secondary metabolites present in all terrestrial plants. The flavonoid pathway has been extensively studied, and many of the involved genes and metabolites have been described in the literature. Despite this extensive knowledge, the functioning of the pathway in vivo is still poorly understood. Here, we study the flavonoid pathway using both experiments and mathematical models. We measured flavonoid metabolite dynamics in two tissues, hypocotyls and cotyledons, during tomato seedling development. Interestingly, the same backbone of interactions leads to very different accumulation patterns in the different tissues. Initially, we developed a mathematical model with constant enzyme concentrations that described the metabolic networks separately in both tissues. This model was unable to fit the measured flavonoid dynamics in the hypocotyls, even if we allowed unrealistic parameter values. This suggested us to investigate the effect of transcript abundance on flavonoid accumulation. We found that the expression of candidate flavonoid genes varies considerably with time. Variation in transcript abundance results in enzymatic variation, which could have a large effect on metabolite accumulation. Candidate transcript abundance was included in the mathematical model as representative for enzyme concentration. We fitted the resulting model to the flavonoid dynamics in the cotyledons, and tested it by applying it to the data from hypocotyls. When transcript abundance is included, we are indeed able to explain flavonoid dynamics in both tissues. Importantly, this is possible under the biologically relevant restriction that the enzymatic properties estimated by the model are conserved between the tissues.


Relationships among reproductive morphology, behavior, and testosterone in a natural population of green anole lizards.
Laboratory studies of reproductive systems have long supported the idea that neural and/or muscular structures used frequently are often enhanced in size. However, field studies integrating behavioral, morphological, and hormonal data are needed to better understand relationships in natural environments. We examined a natural population of green anole lizards (Anolis carolinensis) to determine whether variation in reproductive morphology both within and between the sexes paralleled differences in courtship and copulatory behaviors and circulating testosterone levels. Display rate in males was positively correlated with the sizes of the cartilage supporting the dewlap (a throat fan used in courtship and aggression) and renal sex segments (portions of the kidney that function similarly to the mammalian prostate), but correlated negatively with seminiferous tubule size. Plasma testosterone in males was negatively correlated with display behavior and was not correlated with any measures of morphology. Females, which display rarely, exhibited no relationships between morphology and frequency of behavior. Comparisons between the sexes show that males have consistently larger courtship and copulation morphologies than females, even when accounting for sex differences in body size. The results not only support the idea of relationships between increased function and enhanced structures, but also show the complexity of mechanistic interactions associated with reproductive behavior in wild animals.


Cor Triatriatum Dexter: A Case Report in a 70-Year-Old Male.
Cor triatriatum dexter (CTD) is a rare congenital heart disease resulting from persistence of the right valve of the sinus venosus. The persistent valve forms a membrane that divides the right atrium into a proximal and a distal chamber. This disorder exhibits varying clinical manifestations depending on the degree of partitioning or septation of the right atrium. In asymptomatic patients, the disease may be discovered during surgical procedures, diagnostic testing such as echocardiography, or hemodynamic monitoring. Severe septation abnormalities may cause right-sided heart failure and elevated central venous pressures due to obstruction of the tricuspid valve, right ventricular outflow tract, or inferior vena cava. Here we report a case of CTD in a patient presenting with symptoms of dyspnea and fatigue, followed by a short discussion of the embryology and clinical implications of this congenital disease, as well as current advances in management.


Dynamics of free surface perturbations along an annular viscous film.
It is known that the free surface of an axisymmetric viscous film flowing down the outside of a thin vertical fiber under the influence of gravity becomes unstable to interfacial perturbations. We present an experimental study using fluids with different densities, surface tensions, and viscosities to investigate the growth and dynamics of these interfacial perturbations and to test the assumptions made by previous authors. We find that the initial perturbation growth is exponential, followed by a slower phase as the amplitude and wavelength saturate in size. Measurements of the perturbation growth for experiments conducted at low and moderate Reynolds numbers are compared to theoretical predictions developed from linear stability theory. Excellent agreement is found between predictions from a long-wave Stokes flow model [Craster and Matar, J. Fluid Mech. 553, 85 (2006)] and data, while fair to excellent agreement (depending on fiber size) is found between predictions from a moderate-Reynolds-number model [Sisoev, Chem. Eng. Sci. 61, 7279 (2006)] and data. Furthermore, we find that a known transition in the longer-time perturbation dynamics from unsteady to steady behavior at a critical flow rate Q(c) is correlated with a transition in the rate at which perturbations naturally form along the fiber. For Q<Q(c) (steady case), the rate of perturbation formation is constant. As a result, the position along the fiber where perturbations form is nearly fixed, and the spacing between consecutive perturbations remains constant as they travel 2 m down the fiber. For Q>Q(c) (unsteady case), the rate of perturbation formation is modulated. As a result, the position along the fiber where perturbations form oscillates irregularly, and the initial speed and spacing between perturbations varies, resulting in the coalescence of neighboring perturbations further down the fiber.


The importance of problem formulations in risk assessment: a case study involving dioxin-contaminated soil.
The need to remediate contaminated soils is typically accomplished by applying standard risk assessment methods followed by risk management to select remedial options. These human health risk assessments (HHRAs) have been largely conducted in a formulaic manner that relies heavily on standard deterministic exposure, toxicity assumptions and fixed mathematical formulas. The HHRA approach, with its traditional formulaic practice, does not take advantage of problem formulation in the same manner as is done in ecological risk assessment, and historically, has generally failed to emphasize incorporation of site-specific information. In response to these challenges, the National Academy of Sciences recently made several recommendations regarding the conduct of HHRAs, one of which was to begin all such assessments with problem formulation. These recommendations have since been extended to dose response assessment. In accordance with these recommendations, a group of experts presented and discussed findings that highlighted the importance and impact of including problem formulation when determining the need for remediation of dioxin contamination in soils, focusing in particular on exposure assessment is described.


Growth hormone and prolactin-staining tumors causing acromegaly: a retrospective review of clinical presentations and surgical outcomes.
Acromegaly results in disfiguring growth and numerous medical complications. This disease is typically caused by growth hormone (GH)-secreting pituitary adenomas, which are treated first by resection, followed by radiation and/or medical therapy if needed. A subset of acromegalics have dual-staining pituitary adenomas (DSPAs), which stain for GH and prolactin. Presentations and treatment outcomes for acromegalics with DSPAs are not well understood. The authors retrospectively reviewed the records of more than 5 years of pituitary adenomas resected at their institution. Data were collected on variables related to clinical presentation, tumor pathology, radiological size, and disease recurrence. The Fisher's exact test, ANOVA, Student t-test, chi-square test, and Cox proportional hazards and multiple logistic regression were used to measure statistical significance. Of 593 patients with pituitary adenoma, 91 presented with acromegaly. Of these 91 patients, 69 (76%) had tumors that stained for GH only (single-staining somatotrophic adenomas [SSAs]), while 22 (24%) had tumors that stained for GH and prolactin (DSPAs). Patients with DSPAs were more likely to present with decreased libido (p = 0.012), signs of acromegalic growth (p = 0.0001), hyperhidrosis (p = 0.0001), and headaches (p = 0.043) than patients with SSAs. DSPAs presented with significantly higher serum prolactin (60.7 vs 10.0 µg/L, p = 0.0002) and insulin-like growth factor-1 (IGF-1) (803.6 vs 480.0 ng/ml, p = 0.0001), and were more likely to have IGF-1 levels > 650 ng/ml (n = 13 [81.3%] vs n = 6 [21.4%], p = 0.0001) than patients with SSAs despite similar sizes (1.8 vs 1.7 cm, p = 0.5). Patients with DSPAs under 35 years of age were more likely to have a recurrence (n = 4 [50.0%] vs n = 3 [11.1%], p = 0.01) than patients with SSAs under the age of 35. DSPA patients were less likely to achieve remission with surgery than SSA patients (n = 2 [20%] vs n = 19 [68%], p = 0.01). Univariate analysis identified single-staining tumors (p = 0.02), gross-total resection (p = 0.02), and tumor diameter (p = 0.05) as predictors of surgical remission. Multiple logistic regression demonstrated that SSAs (p = 0.04) were independently associated with surgical remission of acromegaly. Kaplan-Meier analysis revealed that DSPAs had more time until disease remission (p = 0.033). Acromegalics with tumors that stain for prolactin and GH, which represented almost a quarter of acromegalics in this cohort, had more aggressive clinical presentations and postoperative outcomes than SSAs. Prolactin staining provides useful information for acromegalics undergoing pituitary surgery.


Distinct docking and stabilization steps of the Pseudopilus conformational transition path suggest rotational assembly of type IV pilus-like fibers.
The closely related bacterial type II secretion (T2S) and type IV pilus (T4P) systems are sophisticated machines that assemble dynamic fibers promoting protein transport, motility, or adhesion. Despite their essential role in virulence, the molecular mechanisms underlying helical fiber assembly remain unknown. Here, we use electron microscopy and flexible modeling to study conformational changes of PulG pili assembled by the Klebsiella oxytoca T2SS. Neural network analysis of 3,900 pilus models suggested a transition path toward low-energy conformations driven by progressive increase in fiber helical twist. Detailed predictions of interprotomer contacts along this path were tested by site-directed mutagenesis, pilus assembly, and protein secretion analyses. We demonstrate that electrostatic interactions between adjacent protomers (P-P+1) in the membrane drive pseudopilin docking, while P-P+3 and P-P+4 contacts determine downstream fiber stabilization steps. These results support a model of a spool-like assembly mechanism for fibers of the T2SS-T4P superfamily.


A retrospective analysis of environmental risk factors for the diagnosis of deep stromal abscess in 390 horses in North Central Florida from 1991 to 2013.
The purpose of this investigation was to identify potential environmental risk factors for the diagnosis of equine deep stromal abscesses (DSA) in the subtropical climate at the University of Florida Veterinary Medical Center (UFVMC). Cases included were selected from the UFVMC medical record and imaging database, and included all cases of equine DSA diagnosed during the period from December 1991 to December 2013 in patients residing in north central Florida. Patient date of diagnosis and atmospheric data was obtained for north central Florida for the corresponding time period. Univariate and multivariate general linear models were generated testing effects and interactions between environmental conditions. When year, sulfur dioxide (SO2 ) and wind were analyzed in the presence of each other, a one-mile per hour increase in wind (P = 0.005) significantly increased the number of DSA cases by 1.63 cases per year. When the influence of temperature was evaluated in conjunction with year and nitrogen dioxide (NO2 ), the number of cases decreased by 0.1534 per year for every degree increase in temperature (°C) (P = 0.039). Wind speed is the first significant atmospheric risk factor to be identified for DSA formation in the horse. The importance of environmental variance in the incidence of DSA indicates that the pathogenesis of DSA formation may be multifactorial, interdependent and provides support in some horses for the micropuncture hypothesis of DSA formation related to the involvement of environmental conditions causing precorneal tear film and epithelial damage.


High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.
Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.


Drosophila provides rapid modeling of renal development, function, and disease.
The evolution of specialized excretory cells is a cornerstone of the metazoan radiation, and the basic tasks performed by Drosophila and human renal systems are similar. The development of the Drosophila renal (Malpighian) tubule is a classic example of branched tubular morphogenesis, allowing study of mesenchymal-to-epithelial transitions, stem cell-mediated regeneration, and the evolution of a glomerular kidney. Tubule function employs conserved transport proteins, such as the Na(+), K(+)-ATPase and V-ATPase, aquaporins, inward rectifier K(+) channels, and organic solute transporters, regulated by cAMP, cGMP, nitric oxide, and calcium. In addition to generation and selective reabsorption of primary urine, the tubule plays roles in metabolism and excretion of xenobiotics, and in innate immunity. The gene expression resource FlyAtlas.org shows that the tubule is an ideal tissue for the modeling of renal diseases, such as nephrolithiasis and Bartter syndrome, or for inborn errors of metabolism. Studies are assisted by uniquely powerful genetic and transgenic resources, the widespread availability of mutant stocks, and low-cost, rapid deployment of new transgenics to allow manipulation of renal function in an organotypic context.


A new genetic locus controlling growth and proliferation in Drosophila melanogaster.
Multicellular organisms grow through both proliferation and growth of their individual cells. We have conducted a P-element-based misexpression screen for genes whose upregulation alters wing disc growth during development. One particular group of four P elements, all inserted at cytological location 61C7-8, exhibited specific overgrowth upon misexpression in proliferating imaginal tissues. Clonal analysis revealed that upon misexpression, cell number was increased but cell size was not affected, indicating that cell growth and proliferation were induced in a coordinate manner. Loss of function at the locus produced small flies with reduced cell number, consistent with the presence of a gene encoding a positive growth regulator. We characterized a new transcription unit initiating in a region adjacent to the P insertions, which generated a complex series of polyadenylated transcripts. Although these RNAs were induced in response to misexpression, none was sufficient by itself to recapitulate overgrowth when overexpressed. This suggested either that a particular combination of these transcripts was necessary or that other sequences are involved.